Dr. David Lakey completed a Pediatric and Adult Infectious Diseases fellowship at Vanderbilt University Medical Center. He spent 2 years in Dr. Douglas Kernodle's laboratory, studying expression of mycobacterial genes in E. coli. Dr. Lakey received 1 of only 3 fellowships awarded by the Pediatric Infectious Disease Society and was the 1998 Grant Liddle Scholar, given to the best research fellow in the Vanderbilt University Department of Medicine. Dr. Lakey has a superb grasp of molecular microbiology. His ability to think independently, use novel approaches to solve problems, and interact with other scientists make him an excellent candidate for a career in biomedical research. The UT Health Center at Tyler has a long-term commitment to excellence in tuberculosis research, and 5 NIH-funded investigators there are currently engaged in research in mycobacterial disease. The institution is committed to Dr. Lakey's career development and has provided him with a fully equipped laboratory and support staff. Dr. Peter Barnes, an accomplished investigator in tuberculosis, will serve as Dr. Lakey's mentor, and an Advisory Committee of 6 other experienced scientists will provide guidance to Dr. Lakey. Dr. Lakey will devote 90 percent effort to this proposal. The research project is to identify genes of M. tuberculosis that contribute to enhanced mycobacterial growth in macrophages. This information will facilitate development of attenuated M. tuberculosis strains as vaccines, as well as design of drugs for treatment and prevention of tuberculosis. Our specific aims are: 1) To establish methods to quantitate mRNA expression of intracellular mycobacteria, using competitive RT-PCR; 2) To evaluate differential gene expression of laboratory and clinical M. tuberculosis strains during intracellular growth, using competitive RT-PCR, differential display-PCR and mycobacterial microarrays. We will identify genes that are differentially expressed by M. tuberculosis strains that grow at different rates in macrophages; 3) To determine the functional effects of differentially expressed genes identified in aim 2 on intracellular mycobacterial growth. Candidate mycobacterial genes associated with rapid intracellular growth will be expressed by E. coli and by M. tuberculosis H37Ra to assess their effects on intracellular growth. In addition to this research project, Dr. Lakey will take didactic courses offered by the Master's Program in Biotechnology, and participate in other educational activities within and outside of the institution.